scholarly journals Mapping global changes in nuclear cytosine base modifications in the early mouse embryo

Reproduction ◽  
2016 ◽  
Vol 151 (2) ◽  
pp. 83-95 ◽  
Author(s):  
Y Li ◽  
Michelle K Y Seah ◽  
C O'Neill
Keyword(s):  
Dna Methyltransferase ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Early Embryo ◽  
Normal Embryo ◽  
Global Changes ◽  
Cell Cycles ◽  
Early Mouse

Reprogramming epigenetic modifications to cytosine is required for normal embryo development. We used improved immunolocalization techniques to simultaneously map global changes in the levels of 5′-methylcytosine (5meC) and 5′-hydroxymethylcytosine (5hmC) in each cell of the embryo from fertilization through the first rounds of cellular differentiation. The male and female pronuclei of the zygote showed similar staining levels, and these remained elevated over the next three cell cycles. The inner cells of the morula showed a progressive reduction in global levels of both 5meC and 5hmC and further losses occurred in the pluripotent inner cell mass (ICM) of the blastocyst. This was accompanied by undetectable levels of DNA methyltransferase of each class in the nuclei of the ICM, while DNA methyltransferase 3B was elevated in the hypermethylated nuclei of the trophectoderm (TE). Segregation of the ICM into hypoblast and epiblast was accompanied by increased levels in the hypoblast compared with the epiblast. Blastocyst outgrowth in vitro is a model for implantation and showed that a demethylated state persisted in the epiblast while the hypoblast had higher levels of both 5meC and 5hmC staining. The high levels of 5meC and 5hmC evident in the TE persisted in trophoblast and trophoblast giant cells after attachment of the blastocyst to the substratum in vitro. This study shows that global cytosine hypomethylation and hypohydroxymethylation accompanied the formation of the pluripotent ICM and this persisted into the epiblast after blastocyst outgrowth, and each differentiated lineage formed in the early embryo showed higher global levels of 5meC and 5hmC.

In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts

Development ◽  
1978 ◽  
Vol 45 (1) ◽  
pp. 93-105
Author(s):  
Brigid Hogan ◽  
Rita Tilly
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Epithelial Layer ◽  
Visceral Endoderm ◽  
Limited Region ◽  
In Vitro Development ◽  
Vitro Development ◽  
Embryonic Ectoderm

This paper describes the in vitro development of inner cell masses isolated immunosurgically from mouse blastocysts which had been collected on 3·5 days p.c. and then incubated for 24 h. The inner cell masses continue to grow in culture and develop through a series of stages with increasing complexity of internal organization. By day 1 all of the cultured ICMs have an outer layer of endoderm, and by day 3 some of them have two distinct kinds of inside cells; a columnar epithelial layer and a thin hemisphere of elongated cells. Later, mesodermal cells appear to delaminate from a limited region of the columnar layer, close to where it forms a junction with the thinner cells. By day 5, about 25% of the cultured ICMs have a striking resemblance to normal 7·5-day p.c. C3H embryos, with embryonic ectoderm, extra-embryonic ectoderm and chorion, embryonic and extra-embryonic mesoderm, and visceral endoderm. When mechanically disrupted and grown as attached clumps of cells in a tissue dish, these embryo-like structures give rise to trophoblast-like giant cells. These results suggest that the inner cell mass of 4·5-day p.c. blastocysts contains cells which can give rise to trophoblast derivates in culture.

Morphology, developmental stages and quality parameters of in vitro-produced equine embryos

2019 ◽  
Vol 31 (12) ◽  
pp. 1758 ◽  
Author(s):  
Elaine M. Carnevale ◽  
Elizabeth S. Metcalf
Keyword(s):  
Embryo Development ◽  
Developmental Stages ◽  
Inner Cell Mass ◽  
Early Stage ◽  
Cell Mass ◽  
Quality Parameters ◽  
Early Embryo ◽  
Developmental Competence ◽  
Limited Information

Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning of the zona pellucida (ZP) or capsule formation. Cells of the inner cell mass (ICM) are dispersed, in contrast with the differentiated trophoblast and ICM observed in embryos collected from uteri. As blastocysts expand invitro, embryo cells often escape the ZP as organised or disorganised extrusions of cells, probably through the hole incurred during ICSI. Quality assessment of invitro-produced early stage equine embryos is in its infancy, because limited information is available regarding the relationship between morphology and developmental competence. Early embryo development invivo is reviewed in this paper, with comparisons made to embryo development invitro and clinical assessments from a laboratory performing commercial ICSI for >15 years.

scholarly journals Oct4 regulates embryonic pluripotency via metabolic mechanisms and Stat3 signalling

2020 ◽  
Author(s):  
Giuliano Giuseppe Stirparo ◽  
Agata Kurowski ◽  
Stanley Eugene Strawbridge ◽  
Hannah Stuart ◽  
Thorsten Edwin Boroviak ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Specific Gene ◽  
List Type ◽  
Early Mouse

AbstractOCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 protects the pluripotent lineage from differentiation into trophoblast, we used single cell transcriptomics and quantitative immunofluorescence on blastocysts and established differentially expressed genes and pathways between control and OCT4 null cells. Activation of most pluripotency-associated transcription factors in the early mouse inner cell mass appears independent of OCT4, whereas JAK/STAT signalling requires OCT4, via activation of IL6ST. Single cell deconvolution, diffusion component and trajectory inference dissected the process of differentiation of OCT4 null cells by activating specific gene-network and transcription factors. Downregulation of glycolytic and oxidative metabolism was observed. CHIPseq analysis suggests OCT4 directly targets rate-limiting glycolytic enzymes. Concomitant with significant disruption of the STAT3 pathway, oxidative respiration is significantly diminished in OCT4 null cells. Upregulation of the lysosomal pathway detected in OCT4 null embryos is likely attributable to aberrant metabolism.Highlights and noveltyMajor pluripotency-associated transcription factors are activated in OCT4-deficient early mouse ICM cells, coincident with ectopic expression of trophectoderm markersJAK/STAT signalling is defective in OCT4 null embryosOCT4 promotes expression of KATS enzymes by means of glycolytic production of Acetyl CoA to secure chromatin accessibility for acquisition of epiblast identityOCT4 regulates the metabolic and biophysical processes required for establishment of embryonic pluripotency

scholarly journals MCRS1 is essential for epiblast development during early mouse embryogenesis

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Wei Cui ◽  
Agnes Cheong ◽  
Yongsheng Wang ◽  
Yuran Tsuchida ◽  
Yong Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Histone H4 ◽  
Histone H4 Acetylation ◽  
Early Mouse

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO

2009 ◽  
Vol 21 (9) ◽  
pp. 63
Author(s):  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Pluripotent Cells ◽  
Drug Induced ◽  
Developmental Potential

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

In vivo and in vitro development of mouse embryos homozygous for the embryonic lethal velvet coat (Ve) mutation

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 43-55
Author(s):  
J. Rossant ◽  
K. M. Vijh
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Parietal Endoderm ◽  
Trophoblast Giant Cells ◽  
Vitro Development ◽  
Ectoplacental Cone

Embryos homozygous for the velvet coat mutation, Ve/Ve, were recognized at 6·5 days post coitum by the reduced size of the ectodermal portions of the egg cylinder and the loose, columnar nature of the overlying endoderm. Later in development ectoderm tissues were sometimes entirely absent. Abnormalities appeared in the ectoplacental cone at 8·5 days but trophoblast giant cells and parietal endoderm appeared unaffected. Homozygotes could not be unequivocally identified at 5·5 days nor at the blastocyst stage but were recognized in blastocyst outgrowths by poor development of the inner cell mass derivatives, It has previously been suggested that Ve may exert its action at the blastocyst stage by reducing the size of the inner cell mass, but no evidence for such a reduction was found. Most of the observations on Ve/Ve homozygotes are, however, consistent with the hypothesis that Ve exerts its action primarily on later primitive ectoderm development.

Preferential paternal X inactivation in extraembryonic tissues of early mouse embryos

Development ◽  
1982 ◽  
Vol 67 (1) ◽  
pp. 127-135
Author(s):  
Mary I. Harper ◽  
Mandy Fosten ◽  
Marilyn Monk
Keyword(s):  
X Chromosome ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Phosphoglycerate Kinase ◽  
X Inactivation ◽  
Preferential Expression ◽  
Trophoblastic Cells ◽  
Extraembryonic Membranes ◽  
Early Mouse

The preferential expression of the maternal X chromosome seen in certain extraembryonic membranes of the mouse was studied by investigating the tissues from which these membranes are derived during early development. The electrophoretic variant of the X-coded enzyme PGK-1 (phosphoglycerate kinase) was used to distinguish the expression of the maternal from the paternal X chromosome in heterozygous females. Both the extraembryonic ectoderm and primary endoderm of 6½-day female egg cylinders gave almost exclusive expression of the maternal form of the enzyme whereas the epiblast gave near equal expression of the two parental alleles. No paternal PGK-1 band could be detected in samples of pooled 3½-day blastocysts, but after 3 or 4 days of culture in vitro a faint paternal band was seen in the resultant outgrowths. The activity of the maternal band in these latter samples had increased greatly from that of the blastocysts, consistent with preferential expression of the maternal Pgk-1 allele in the trophoblastic cells of the outgrowths, while both alleles are expressed in inner-cell-mass cells. The results strongly support the idea that non-random X-chromosome expression is due to preferential paternal X inactivation in trophectoderm (from which extraembryonic ectoderm is derived) and in primary endoderm, and not to cell selection.

In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts

Development ◽  
1978 ◽  
Vol 45 (1) ◽  
pp. 107-121
Author(s):  
Brigid Hogan ◽  
Rita Tilly
Keyword(s):  
Outer Layer ◽  
Normal Mouse ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Visceral Endoderm ◽  
Floating Structures ◽  
Parietal Endoderm ◽  
Vitro Development

This paper describes the development in culture of inner cell masses isolated immunosurgically from C3H/He mouse blastocysts immediately after collection between 3·5 and 4·0 days p.c. By 24–48 h most of the inner cell masses isolated from half-expanded blastocysts, and about 50% of those from expanded blastocysts, regenerate an outer layer of trophectoderm- like cells and so resemble mini-blastocysts. With further in vitro culture these structures attach to the substratum and give rise to trophoblast-like giant cells, together with clusters of parietal endoderm cells or inner cell masses surrounded by visceral endoderm. Many of the inner cell masses from the remaining expanded blastocysts develop into floating structures with an outer layer of endoderm cells, and by 7 days consist of a large fluid filled cyst surrounding a collapsed vesicle of epithelial cells. Mesodermal cells line the cysts and form numerous blood islands. When mechanically disrupted, and grown as attached sheets of cells, these cystic structures give rise to patches of trophoblast-like giant cells similar to those described in the previous paper. These results suggest that the inner cell mass of normal mouse blastocysts contains cells which are capable of giving rise to trophoblast in culture.

Determinants of valid measurements of global changes in 5ʹ-methylcytosine and 5ʹ-hydroxymethylcytosine by immunolocalisation in the early embryo

2015 ◽  
Vol 27 (5) ◽  
pp. 755 ◽  
Author(s):  
J. Salvaing ◽  
Y. Li ◽  
N. Beaujean ◽  
C. O'Neill
Keyword(s):  
Cytosine Methylation ◽  
Inner Cell Mass ◽  
Low Cost ◽  
Classical Model ◽  
Cell Mass ◽  
Early Embryo ◽  
Global Changes ◽  
Cpg Dinucleotides ◽  
Base Level ◽  
Stage Of Development

A classical model of epigenetic reprogramming of methyl-cytosine–phosphate–guanine (CpG) dinucleotides within the genome of the early embryo involves a process of active demethylation of the paternally derived genome immediately following fertilisation, creating marked asymmetry in global cytosine methylation levels in male and female pronuclei, followed by passive demethylation of the maternally derived genome over subsequent cell cycles. This model has dominated thinking in developmental epigenetics over recent decades. Recent re-analyses of the model show that demethylation of the paternally derived genome is more modest than formerly thought and results in overall similar levels of methylation of the paternal and maternal pronuclei in presyngamal zygotes, although there is little evidence for a pervasive process of passive demethylation during the cleavage stage of development. In contrast, the inner cell mass of the blastocyst shows some loss of methylation within specific classes of loci. Improved methods of chemical analysis now allow global base-level analysis of modifications to CpG dinucleotides within the cells of the early embryo, yet the low cost and convenience of the immunolocalisation techniques mean that they still have a valuable place in the analysis of the epigenetics of embryo development. In this review we consider the key strengths and weaknesses of this methodology and some factors required for its valid use and interpretation.

scholarly journals mTOR Is Essential for Growth and Proliferation in Early Mouse Embryos and Embryonic Stem Cells

2004 ◽  
Vol 24 (15) ◽  
pp. 6710-6718 ◽  
Author(s):  
Mirei Murakami ◽  
Tomoko Ichisaka ◽  
Mitsuyo Maeda ◽  
Noriko Oshiro ◽  
Kenta Hara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Size ◽  
Inner Cell Mass ◽  
Critical Role ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Mouse Embryos ◽  
Early Mouse

ABSTRACT TOR is a serine-threonine kinase that was originally identified as a target of rapamycin in Saccharomyces cerevisiae and then found to be highly conserved among eukaryotes. In Drosophila melanogaster, inactivation of TOR or its substrate, S6 kinase, results in reduced cell size and embryonic lethality, indicating a critical role for the TOR pathway in cell growth control. However, the in vivo functions of mammalian TOR (mTOR) remain unclear. In this study, we disrupted the kinase domain of mouse mTOR by homologous recombination. While heterozygous mutant mice were normal and fertile, homozygous mutant embryos died shortly after implantation due to impaired cell proliferation in both embryonic and extraembryonic compartments. Homozygous blastocysts looked normal, but their inner cell mass and trophoblast failed to proliferate in vitro. Deletion of the C-terminal six amino acids of mTOR, which are essential for kinase activity, resulted in reduced cell size and proliferation arrest in embryonic stem cells. These data show that mTOR controls both cell size and proliferation in early mouse embryos and embryonic stem cells.

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